17-Azolyl steroids useful as androgren synthesis inhibitors

ABSTRACT

Androgen synthesis inhibitors, as well as methods for the use of the same to reduce plasma levels of testosterone and/or dyhydrotestosterone, and to treat prostate cancer and benign prostatic hypertrophy, are disclosed.

This is a divisional of application Ser. No. 08/953,403 filed Oct. 17,1997.

The development of the present invention was supported by the Universityof Maryland, Baltimore, Md. and by funding from the National Institutesof Health under grant number CA 27440. The United States Government hasa non-exclusive, irrevocable, paid-up license to practice or havepracticed for or on behalf of the United States the invention herein asprovided for by the terms of the above mentioned contracts awarded bythe United States Government.

FIELD OF THE INVENTION

The present invention relates to novel 17-azolyl steroids which areuseful as androgen synthesis inhibitors, as well as methods for the useof the same to reduce plasma levels of testosterone and/ordyhydrotestosterone, and to treat prostate cancer and benign prostatichypertrophy.

BACKGROUND OF THE INVENTION

Breast cancer kills 45,000 women per year. In addition, prostate cancernow ranks as the most prevalent cancer in men. Approximately 160,000 newcases are diagnosed with prostate cancer each year. Of these, 35,000will die of metastatic disease.

It has been proposed that selective aromatase (estrogen synthetase)inhibitors to control estrogen production would be useful agents fortreatment of breast cancer in women (Bolla et al, N. Eng. J. Med.,337:295-300 (1997)). In addition, in men, aromatase inhibitors may beuseful for conditions associated with estrogen excess, such asgynecomastia and oligospermia (Coen et al, New Eng. J. Med., 324:317-322(1991); and Hsiang et al, J. Steroid Biochem., 26:131-136 (1987)). Ithas also been suggested that aromatase inhibitors might be useful in thetreatment of prostatic cancer and benign prostatic hypertrophy (BPH)(Henderson, Annals Med., 23:201-203 (1991)).

Compounds which are potent and selective inhibitors of aromatase havebeen reported (Schwarzel et al, Endocrinol., 92:866-880 (1973)). Themost active of those inhibitors, 4-hydroxyandrostene-3,17-dione (4-OHA)(Brodie et al, J. Steroid Biochem., 7:787-793 (1976)), was found to actby rapid competitive inhibition, followed by inactivation of the enzymein vitro, which appeared to be long-lasting or irreversible (Brodie etal, Steroids, 38:693-702 (1981)). Enzyme inhibitors with theseproperties are thought to bind to the active site of the enzyme, arelikely to be quite specific, and should have long-lasting effects invivo due to inactivation of the enzyme (Sjoerdsma, Clin. Pharmacol.Ther., 30:3 (1981)). It was also demonstrated that 4-OHA reducesperipheral plasma estrogen levels, and causes significant regression ofbreast cancers in postmenopausal patients with advanced metastaticdisease who have relapsed from other hormonal treatment, such asovariectomy and tamoxifen. 4-OHA has both oral and parenteral activity,and is without significant side-effects in these patients (Goss et al,Cancer Res., 46:4223-4826 (1986); and Coombes et al, Steroids,50:245-252 (1987)). 4-OH-A, also known as formastane, was approved in1995 for the treatment of breast cancer in many countries worldwide,including most European countries and Canada. It was the first newtreatment for breast cancer in 10 years.

In men, estrogens are produced by the testes, and by peripheralaromatization of adrenal androgens. Testosterone is the major product ofthe testis, and is converted in the prostate by 5α-reductase to the morepotent androgen, dihydrotestosterone (DHT) (Bruchovsky et al, J. Biol.Chem., 243:2012-2021 (1968)). While androgens are of primary importancein the growth of normal prostate, BPH and prostatic cancer, severallines of evidence suggest that estrogens may also have a role (Mawhinneyet al, Adv. Sex Horm. Res., 2:41-209 (1976)).

4-OHA also inhibits 5a-reductase in vitro, although with less potencythan it inhibits aromatase (Brodie et al, Cancer Res., 49:6551-6555(1989b)). Because of these two activities, the possibility that 4-OHAmight be effective in prostatic cancer was explored in a small group ofmen with advanced disease. Subjective responses were observed in 80% ofthese patients, although there was no clear evidence of objectiveremissions (Shearer et al, In: 4-hydroxyandrostenedione—A New Approachto Hormone-Dependent Cancer, Eds. Coombes et al, pages 41-44 (1991)).Estrogen levels were reduced as expected but, DHT concentrations wereunchanged in the patients. The latter finding, in addition to the weakandrogenic activity of 4-OHA, may have determined the lack of objectiveresponses.

Chemotherapy is usually not highly effective, and is not a practicaloption for most patients with prostatic cancer because of the adverseside-effects which are particularly detrimental in older patients.However, the majority of patients initially respond to hormone ablativetherapy (McGuire, In: Hormones and Cancer, Eds. Iacobelli et al, RavenPress, New York, Vol. 15, pages 337-344 (1980)) although they eventuallyrelapse, as is typical with all cancer treatments. Current treatment byorchidectomy or administration of gonadotropin-releasing hormone (GnRH)agonists result in reduced androgen production by the testis, but doesnot interfere with androgen synthesis by the adrenals. Following 3months of treatment with a GnRH agonist, testosterone and DHTconcentrations in the prostate remained at 25% and 10%, respectively, ofpretreatment levels (Forti et al, J. Clin. Endocrinol. Metab.,68:461-468 (1989)). Similarly, about 20% of castrated patients inrelapse had significant levels of DHT in their prostatic tissue (Gelleret al, J. Urol., 132:693-696 (1984)). These finding suggest that theadrenals contribute precursor androgens to the prostate. This issupported by clinical studies of patients receiving combined treatmentwith either GnRH or orchidectomy and an anti-androgen, such asflutamide, to block the actions of androgens, including adrenalandrogens. Such patients have increased progression-free survival timecompared to patients treated with GnRH agonist or orchidectomy alone(Crawford et al, N. Engl. J. Med., 321:419-424 (1989); and Labrie et al,Cancer Suppl., 71:1059-1067 (1993)).

Although patients initially respond to endocrine therapy, theyfrequently relapse. It was reported recently that in 30% of recurringtumors of patients treated with endocrine therapy, high-level androgenreceptor (AR) amplification was found (Visakorpi et al, Nature Genetics,9:401-406 (1995)). Also, flutamide tends to interact with those mutantAR, and stimulate prostatic cell growth. This suggests that ARamplification may facilitate tumor cell growth in low androgenconcentrations. Thus, total androgen blockade as first line therapy maybe more effective than conventional androgen deprivation by achievingmaximum suppression of androgen concentrations which may also prevent ARamplification. It is presently unclear whether sequential treatment withdifferent agents can prolong the benefits of the initial therapy. Thisstrategy has been found effective in breast cancer treatment. New agentswhich act by different mechanisms could produce second responses in aportion of relapsed patients. Although the percentage of patients whorespond to second-line hormonal therapy may be relatively low, asubstantial number of patients may benefit because of the high incidenceof prostatic cancer. Furthermore, there is the potential for developingmore potent agents than current therapies, none of which are completelyeffective in blocking androgen effects.

Human cytochrome 17α-hydroxylase/C_(17,20)-lyase (hereinafter“P450_(17α)”) is a key enzyme in the biosynthesis of androgens, andconverts the C₂₁ steroids (pregnenolone and progesterone) to the C₁₉androgens, dehydroepiandrosterone (DHEA), 5-androstenediol (A-diol),testosterone, and 5 androstenedione in the testis and adrenals. Someinhibitors of P450_(17α) have been described (Barrie, J. SteroidBiochem., 33:1191-1195 (1989); McCague et al, J. Med. Chem.,33:3050-3055 (1990); Jarman et al, J. Med. Chem., 33:2452-2455 (1990);Ayub et al, J. Steroid Biochem., 28:521-531 (1987); Nakajin et al,Yakugaku Zasshi. (Japan), 108:1188-1195 (1988); Nakajin et al, Chem.Pharm. Bull. (Tokyo), 37:1855-1858 (1989); Angelastro et al, Biochem.Biophys. Res. Commun., 162:1571-1577 (1989); Potter et al, J. Med.Chem., 38:2463-2471 (1995); and Rowlands et al, J. Med. Chem.,38:4191-4197 (1995)). Ketoconazole, an active imidazole fungicide, hasbeen used to reduce testosterone biosynthesis in the treatment ofpatients with advanced prostatic cancer (Trachtenberg, J. Urol.,132:61-63 (1984); and Williams et al, Br. J. Urol., 58:45-51 (1986)).However, ketoconazole is not very potent. Moreover, it has a number ofsignificant side-effects, including inhibition of several othercytochrome P₄₅₀ steroidogenic enzymes, and reduction of cortisolproduction. Another drug used for prostate cancer, aminoglutethimide(AG), has similar drawbacks. This suggest that more potent and selectiveinhibitors of P450_(17α) could provide useful agents in treating thisdisease. In addition such compounds may be effective in treating breastcancer patients. AG was used for this purpose, but was associated withadverse side-effects.

In the prostate, 5α-reductase is the enzyme that converts testosteroneto the more potent androgen, DHT, which stimulates prostatic growth.This enzyme occurs in two important isoforms, the Type I isoformexpressed in human non-genital skin, and the Type II isoform present inthe human prostate (Russell et al, Ann. Rev. Biochem., 63:25-61 (1994)).The 5α-reductase inhibitor,N-[1,1-dimethyl-3-oxo-4-aza-5αandrost-1-ene-17β-carboxamide(finasteride; Merck) recently approved for treatment of BPH (Stoner, J.Steroid Biochem. Molec. Biol., 37:375-378 (1990)) is a more potentinhibitor of the Type II than of the Type I isoform. However,finasteride is effective mainly in BPH patients with minimal disease,possibly because serum DHT levels have been found to be incompletelyreduced (65-80%). As the Type I isoenzyme is probably the source of muchof the residual plasma DHT, compounds that inhibit Type I as well asType II may be more effective in patients.

More recently, another azasteroid, MK-434, has been described whichreduces prostatic DHT levels in dogs more effectively than finasteride(Cohen et al, The Prostate, 26:55-71 (1995)). The main advantage of thiscompound, which has similar activity to finasteride in vitro, appears tobe its more favorable pharmacokinetics. However, its efficacy in humansremains to be seen. Although finasteride and MK-434 reduce DHT levels,they also increase serum testosterone levels (Geller et al, J. Clin.Endocrinol. Metab., 71:1552-1555 (1990). Preservation of testosteronelevels may be an advantage in patients with BPH. However, inhibitors of5α-reductase which increase testosterone levels may not be sufficientlyeffective in treating prostatic cancer since testosterone will bind tothe AR in the absence of DHT. That is, while DHT binds to the AR withhigher affinity than testosterone and dissociates more slowly,testosterone can bind to the AR when DHT levels are reduced (Gormley,Urol. Clinics of North America, 18(1):93-97 (1991)). As indicated above,despite significant reductions in prostatic DHT levels during treatment(Cohen et al, supra) , these compounds are not as effective ascastration. More importantly, it appears that they are less effective ineliciting prostatic cell death. The androgen-responsive gene, TRPM-2associated with apoptosis is significantly enhanced by castration but,not by finasteride treatment (Rittinaster et al, Mol. Endocrin.,5:1023-1029 (1991); and Shao et al J. Androl., 14:79-86 (1993)). Thishas been attributed to the lower androgen levels after castration (Shaoet al, supra), which is mainly a consequence of the reduction intestosterone production. Recent studies of patients receiving long-termtreatment with finasteride found some patients developed gynecomastiawhich led to breast cancer in a few cases (Green et al, Letter to NewEng. J. Med., 335(11):823-C (1996)). This raises concerns about the useof 5α-reductase inhibitors, since blockade of this step increases theconversion of androgen substrates to estrogens. Compounds which reduceproduction of testosterone and DHT as well as other androgens byinhibiting P450_(17α) would not be associated with this problem, and maybe more effective in the treatment of prostatic cancer.

Several compounds which inhibit both P450_(17α) and 5α-reductase havebeen identified (Li et al, J. Steroid Biochem. Mol. Biol., 42:313-321(1992); Li et al, The Prostate, 26:140-150 (1995); and Li et al, J. Med.Chem., 39:4335-5339 (1996)). Such compounds could block all androgensynthesis, i.e., testosterone, DHT and androstenedione, and be moreeffective alternatives or additions to orchiectomy in treating prostatecancer patients.

In pending U.S. patent application Ser. No. 08/795,932, filed Feb. 5,1997; which is incorporated by reference herein in its entirety,compounds which inhibit androgen synthesis have been identified andpurified.

In the present invention, additional compounds which inhibit androgensynthesis have been identified and purified. These compounds stronglyinhibit P450_(17α), and are based on the finding that an imidazolemoiety acts as a ligand to bind the iron atom of the heme prostheticgroup of P450_(17α), and form a coordinated complex. Such compounds arepotent inhibitors of aromatase, e.g., fadrozole, which is useful in thetreatment of breast cancer (Lang et al, J. Steroid Biochem. Molec.Biol., 44:421-428 (1993)). Although the detailed mechanism of the17α-hydroxylation and C_(17,20)-side-chain cleavage by P450_(17α) inpresently unclear, it appears that the C₁₇ and C₂₀ positions of thesubstrate must be close to the heme group of the enzyme. Thus,introduction of an imidazole group or other heterocyclic group with anitrogen lone pair of electrons at these positions might coordinate tothe iron atom of the prosthetic group in the active site of the enzyme(Green et al, supra). Using this rationale, a series of androstenederivatives (substrate-like compounds) with imidazole, pyrazole andoxazole groups substituted at the 17-position were synthesized in thepresent invention.

The compounds of the present invention, wherein the azole group isattached to the steroid nucleus via a nitrogen of the azole constitute aclass of compounds not hitherto reported, and distinguish the compoundsof the present invention from the known 17-azole androstene steriods.

SUMMARY OF THE INVENTION

An object of the present invention is to provide inhibitors of androgenbiosynthesis.

Another object of the present invention is to provide a method for thesynthesis of said inhibitors.

Still another object of the present invention is to providepharmaceutical compositions containing the same.

Yet another object of the present invention is to provide methods forinhibiting synthesis of testosterone and/or DHT.

An additional object of the present invention is to provide methods fortreatment of prostatic cancer and BPH.

These and other objects of the present invention, which will be apparentfrom the detailed description of the invention provided hereinafter,have been met, in one embodiment, by a compound of general Formula (I)or a pharmaceutically acceptable salt thereof:

wherein X represents the residue of the A, B and C rings of a steroidconsisting of a 4-en-3-one or 5-en-3β-ol system;

wherein Az represents an azole group attached to C-17 of the steroid viaa hetero nitrogen atom; and

wherein R₁ and R₂ each represents a hydrogen atom or together representa double bond.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show the schematic synthesis of Compounds 1-30 described inSynthesis Examples 1-25.

FIG. 2A shows a Lineweaver-Burk analysis (1/v vs. i/[s]) ofΔ¹⁶-17-(1H-imidazole) (Compound 11) at 3.0, 6.0 and 10 nM; while FIG. 2Bshows a replot of the slopes of each reciprocal plot versus inhibitorconcentration [I] to obtain the K_(i) value. The inhibition experimentswith the other azoles (Compounds 6, 7, 12, 20 and 21) gave plots thatwere essentially the same as shown herein.

FIG. 3 shows progress curves for the inhibition of human testicularmicrosomal P450_(17α) by Δ⁶-17-(1H-imidazole) (Compound 11) at different25 concentrations.

FIG. 4 shows difference absorption spectra, wherein the experimental andreference cuvettes contained a modified form of P450_(17α) (P450concentration 1.8 μM). The spectra show the effects of addition of 20 μMΔ¹⁶-17-(1H-1,2,3-triazole) (Compound 6), (curve A) and 20 μMΔ¹⁶-17-(1H-imidazole) (Compound 11), (curve B).

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, in one embodiment, the above-described object of thepresent invention have been met by a compound of general Formula (I) ora pharmaceutically acceptable salt thereof.

In Formula (I), X represents the residue of the A, B and C rings of asteroid consisting of 4-en-3-one or 5-en-3β-ol systems; Az represents anazole group attached to C-17 of the steroid via a hetero nitrogen atom;and R₁ and R₂ each represents a hydrogen atom or together represent adouble bond.

Preferred examples of Az include the following groups

The steroid of Formula (I) preferably comprises a basic structureselected from the group consisting of Δ⁴-3-one, Δ⁵-3β-ol andΔ^(1,4)-3-one.

Furthermore, the steroid is preferably an aza steroid comprising a ringnitrogen atom in place of a ring carbon atom, preferably the ringnitrogen is in the A ring, e.g., a 4-azasteroid; or the ring nitrogen isin the B ring, e.g., 6-azasteroid.

Specific examples of the compound of the present invention include thefollowing compounds: 17-(1H-imidazolyl)-androst-5-en-3β-ol,17β-(1H-imidazolyl)-androsta-5,16-dien-3β-ol,17β-(1H-1,2,3-triazolyl)-androst-5-en-3β-ol, 17-(1H-1, 2,3-triazolyl)-androsta-5, 16-dien-3β-ol, 17β-(1H-1,2,4-triazolyl)-androst-5-en-3β-ol,17-(1H-1, 2,4-triazolyl) -androsta-5,16-dien-3β-ol or 3-acetatesthereof; and the following compounds:17β-(1H-imidazolyl)-androst-4-en-3-one,17-(1H-imidazolyl)-androsta-4,16-dien-3-one,17β-(1H-1,2,3-triazolyl)-androst-4-en-3-one, 17-(1H-1, 2, 3-triazolyl)-androsta-4,16-dien-3-one, 17β-(1H-1,2,4-triazolyl)-androst-4-en-3-one,17-(1H-1,2,4-triazolyl)-androsta-4,16-dien-3-one or 3-oximes thereof.

Acetates can be prepared as described in the Synthesis Examples providedherein.

Oximes can be prepared by refluxing the steroids with hydroxylaminehydrochloride in ethanol for 4 hrs, as adding water, and separating thecrude mixture flash chromatography on silica gel.

The particular pharmaceutical acceptable salt of the compounds of thepresent invention is not critical thereto.

Examples of pharmaceutically acceptable base salts which can be used inthe present invention include base salts derived from an appropriatebase, such as alkali metal (e.g., sodium), alkaline earth metal (e.g.,magnesium), ammonium, and NW_(n)H_(m) bases, wherein each of n and m are0 to 4 and n+m is 4, and wherein W is a C₁-C₁₈ alkyl.

Examples of pharmaceutically acceptable salts of an acid group which canbe employed in the present invention include salts of organic carboxylicacids, such as acetic, citric, oxalic, lactic, tartaric, malic,isothionic, lactobionic, ascorbic and succinic acids; organic sulfonicacids, such as methanesulfonic, ethanesulfonic, benzenesulfonic andp-tolylsulfonic acids; and inorganic acids, such as hydrochloric,sulfuric, phosphoric, hydrobromic and sulfamic acids.

The pharmaceutically acceptable salt may be also a salt of an aminogroup, or a 3-hydroxy ester.

Examples of pharmaceutically acceptable salts of an amino group includesalts of the inorganic or strong organic acids noted above.

Examples of pharmaceutically acceptable salts of a hydroxy group includethe anion of the compound in to combination with a suitable cation, suchas Na⁺, and NW_(n)H_(m), wherein W is a C₁-C₁₈ alkyl group, and n and mare 0 to 4, and n+m is 4.

The compounds of Formula (I) can be prepared by a methodology startingfrom the 3β-acetoxyandrost-5-en-17-one represented by Formula (II)(which is commercially available from Aldrich, Milwaukee, Wis.):

This method involves conversion of the compound represented by Formula(II) following the Vilsmeier-Haack reaction (Siddiqui et al, J.Heterocyclic Chem., 32:353-354 (1995)) with phosphorous oxychloride(POCl₃) and dimethylformamide (DMF) to give the3β-acetoxy-17-chloro-16-formylandrosta-5,16-diene represented by Formula(III):

Treatment of the compound represented by Formula (III) with a variety ofazole nucleophiles in (DMF) at 75-80° C. under N₂ atmosphere gives highyields (73-92%) of the 17-azole-Δ¹⁶ steroids represented by Formula(IV):

wherein X represents A, B and C rings. Of the compound represented byFormula (III).

Following decarbonylation at C-16 and cleavage of the 3β-acetoxy group,the 5-en-3β-ol-17-azole compounds of Formula (V) are obtained; whileoppenauer oxidation of these compounds yields their corresponding4-en-3-one-17-azole counterparts.

wherein X is as defined for the compounds of Formula (I).

Analogues of a saturated D-ring can be prepared from the correspondingΔ¹⁶ compounds by reduction with diimide (Potter et al, supra).

A mechanism for the formation of the compound represented by Formula(IV) is outlined in the following reaction scheme; a nucleophilicvinylic “addition-elimination” substitution reaction. Recent studies inthis field favor path a of the Reaction Scheme (Rappoport, Acc. Chem.Res., 25:474-480 (1992)).

It has been found in the present invention that the azoles representedby Formula (I) are potent inhibitors of 17α-hydroxylase-C_(17,20)-Lyase(hereafter referred to as “P450_(17α)”), the enzyme which catalyzes theconversion of progesterone and pregnenolone into the androgens,androstenedione and dehydroepiandrosterone, respectively. Sinceandrogens are implicated in the etiology of a number ofandrogen-dependent diseases, e.g., prostate cancer, inhibitors ofP450_(17α) are useful for the treatment of these diseases. In addition,in has been found in the present invention that some of these potentP450_(17α) inhibitors are also potent inhibitors of 5α-reductase, whilesome have strong anti-androgen activity. Because of their dualactivities, the compounds of the present invention are believed to bemore effective than the current agents in the treatment of prostatecancer and other disease states which depend upon androgens.

Thus, as discussed above, in still another embodiment, theabove-described objects of the present invention have been met by apharmaceutical composition for reducing plasma levels of testosteroneand/or DHT in a mammal in need of such treatment comprising:

(A) a pharmaceutically effective amount of at least one compound ofFormula (I) or a pharmaceutically acceptable salt thereof, and

(B) a pharmaceutically acceptable carrier or diluent.

The compound may be present in the composition in amount of 0.01 to 99.9wt %, and more preferably in about 0.1 to 99 wt % of the composition.Still more preferably, the compound may be present in the composition inan amount of about 1.0 to 70 wt % of the composition.

The particular carrier or diluent employed is not critical to thepresent invention. Typically, the carrier or diluent may be a solid,liquid, or vaporizable carrier, or combinations thereof. Examples ofsuch carriers or diluents include water, ethyl alcohol, propyleneglycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol,polyoxyethylene sorbitol and sorbitate esters. In these instances,adequate amounts of isotonicity adjusters, such as sodium chloride,glucose or glycerin can be added to make the preparations isotonic.Aqueous sterile injection solutions may further contain anti-oxidants,buffers, bacteriostats, and like additives acceptable for parenteralformulations.

In addition, the pharmaceutical composition can contain excipients, suchas fillers, binders, wetting agents, disintegrators, surface-activeagents, lubricants, and the like.

The pharmaceutical composition can be prepared in accordance withaccepted pharmaceutical procedures, for example, as described inRemington's Pharmaceutical Sciences, Seventeenth Edition, ed. Gennaro,Mack Publishing Company, Easton, Pa. (1985).

The composition may be in a unit dosage form. Typical unit dosage formsinclude tablets, pills, powders, solutions, suspensions, emulsions,granules, capsules, suppositories, injectable solutions and suspensions.

As discussed above, in yet another embodiment, the above-describedobjects of the present invention have been met by a method of inhibitingsynthesis, i.e., plasma levels, of testosterone and/or DHT comprisingadministering to a subject in need of such treatment, a pharmaceuticallyeffective amount of at least one compound of Formula (I) or apharmaceutically acceptable salt thereof.

The mode of administering is not critical to the present invention.Examples of the mode of administering include oral, rectal, nasal,topical (including buccal and sublingual), and parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, and transdermal)administration. The preferred modes of administration are oral, nasal,topical and parenteral administration.

The amount of compound of Formula (I) to be administered variesdepending upon the age, weight and species of the subject, the generalhealth of the subject, the severity of the symptoms, whether thecomposition is being administered alone or in combination with othertherapeutic agents, the incidence of side-effects and the like.

In general, a dose suitable for treatment of BPH is about 0.001 to 100mg/kg body weight/dose, preferably about 0.01 to 60 mg/kg bodyweight/dose, and still more preferably about 0.1 to 40 mg/kg bodyweight/dose per day. A dose suitable treatment of prostate cancer isabout 0.001 to 100 mg/kg body weight/dose, preferably about 0.01 to 60mg/kg body weight/dose, and still more preferably about 0.1 to 40 mg/kgbody weight/dose per day. The desired dose may be administered as 1 to 6or more subdoses administered at appropriate intervals throughout theday. The compounds may be administered repeatedly over a period ofmonths or years, or may be slowly and constantly infused to the subject.Higher and lower doses may also be administered.

The daily dose may be adjusted taking into account, for example, theabove-identified variety of parameters. Typically, the compounds ofFormula (I) may be administered in an amount of about 0.001 to 100 mg/kgbody weight/day. However, other amounts may also be administered.

To achieve good plasma concentrations, the compounds may beadministered, for instance, by intravenous injection of an approximate0.1 to 1.0% (w/v) solution of the active ingredient, optionally insaline, or orally administered as a bolus.

In yet another embodiment, the above-described objects of the presentinvention have been met by a method of treating BPH or prostate cancer,or inhibiting the growth of prostate tissue, in a subject in need ofsuch treatment comprising administering a pharmaceutically effectiveamount of the compound of Formula (I) or a pharmaceutically acceptablesalt thereof.

The above-described methods may be practiced by administration of thecompounds by themselves or in a combination with other activeingredients, including other steroid compounds and/or therapeuticagents. Other therapeutic agents suitable for use herein are anycompatible drugs that are effective by the same or other mechanisms forthe intended purpose, or drugs that are complementary to those of thepresent agents. These compounds include agents that are effective forthe inhibition of testosterone and/or DHT synthesis, and in thetreatment of prostate cancer, anticancer agents. Examples of suchcompounds include ketoconazole, finasteride, and 4MA.

The compounds utilized in combination therapy may be administeredsimultaneously, in either separate or combined formulations, or atdifferent times than the present compounds, e.g., sequentially, suchthat a combined effect is achieved. The amounts and regime ofadministration will be adjusted by the practitioner, by preferablyinitially lowering their standard doses and then titrating the resultsobtained.

The following examples are provided for illustrative purposes only, andare in no way intended to limit the scope of the present invention.

In the Synthesis Examples (see FIGS. 1A-1F), ¹H NMR data (300 MHZ)(internal standard Me₄Si=δ0) were recorded on a QE 300, NMR system,General Electric Co., in CDCl₃ unless otherwise stated.

Synthesis reactions were monitored by TLC on silica gel plates (MerckType 60H), and visualized by dipping in 4.0% (v/v) sulfuric acid inethanol followed by heating at about 120-150° C.

Flash column chromatography was carried out on silica gel (Merck grade9385, 230-400 mesh 60 Å) in the solvent systems indicated.

LP refers to petroleum fractions, b.p. 35-60° C.

Solutions were dried using anhydrous Na₂SO₄.

Melting points were measured on a Fischer-Johns Melting Point apparatusand are uncorrected.

Synthesis Example 1

This Example describes a Vilsmeier-Haack reaction of3β-acetoxyandrost-5-en-17-one (Compound 1),3β-acetoxy-17-chloroandrosta-5,16-diene (Compound 2) and3β-acetoxy-17-chloro-16-formylandrosta-5,16-diene (Compound 3).

A solution of 3β-acetoxyandrost-5-en-17-one (Compound 1) (2.0 g, 6.6mmol) in dry chloroform (40 ml) was added dropwise to a cold and stirredsolution of POCl₃ (10 ml) and DMF (10 ml). The mixture was allowed toattain room temperature, and then ref luxed under N₂ for 5 h. It wasthen concentrated under reduced pressure, and poured onto ice followedby extraction with a mixture of ether and EtOAc (8:2 (v/v)). Thecombined extracts were washed with brine, dried (Na₂SO₄), and solventremoved to give a white solid (2.3 g). Analytical TLC (silica gel, pet.ether/EtOAc, (10:1)) revealed the presence of two compounds, both lesspolar than (Compound 1). Purification by flash column chromatography(FCC, silica gel, pet. ether/EtOAc, (15:1)) gave Compound 2 (0.24 g,11.4%) and Compound 3 (1.75 g, 77%).

Analytical and spectroscopic data for Compound 2 and Compound 3 were asfollows:

Compound 2: m.p. 160-162° C.; ¹H NMR (300 MHz, CDCl₃): δ 0.89 (3H, s,18-Me), 1.06 (3H, s, 19-Me), 2.04 (3H, s, 3β-OAc), 4.61 (1H, m, 3α-H),5.39 (1H, d, J=4.8 Hz, 6-H) and 5.63 (1H, d, J=0.9 Hz, 16-H). Analysiscalculated for C₂H₂O₂Cl: C, 72.38; H, 8.39. Found: C, 72.72; H, 8.60.HRMS calcd. for C₂₁H₂₉O₂Cl, 348.1856, found 348.1766.

Compound 3: m.p. 163-165° C.; ¹H NMR (300 MHz, CDCl₃): δ 0.99 (3H, s,18-Me), 1.07 (3H, s, 19-Me), 2.04 (3H, s, 3β-OAc), 4.60 (1H, m, 3a-H),5.40 (1H, d, J=4.8 Hz, 6-H) and 9.99 (1H, s, 16-CHO). Analysiscalculated for C₂H₂O₃Cl: C, 70.11; H, 7.76. Found: C, 70.18; H, 7.82.HRMS calcd. for C₂₁H₂₉O₃Cl, 376.1805, found 376.1748.

Synthesis Example 2

This Example describes the preparation of 3β-acetoxy-17-(1H,1,2,4-triazol-1-yl)-16-formylandrosta-5,16-diene (Compound 4).

A solution of 3β-acetoxy-17-chloro-16-formylandrosta-5,16-diene(Compound 3) (0.6 g, 1.6 mmol) and sodium triazolate (436 mg, 4.79 mmol,3 equiv.) in dry DMF (10 ml) under N₂ was stirred at 78° C. for 30 min.After cooling to room temperature, the reaction mixture was poured ontoice-water (250 ml), and the resulting white precipitate was filtered,washed with water, and dried to give a white solid. This wascrystallized from hexane/EtoAc to give Compound 4 (580 mg, 89%), m.p.160-162° C. ¹H NMR (300 MHz, CDCl₃): δ 1.08 (3H, s, 18-Me), 1.20 (3H, s,19-Me), 2.04 (3β, s, 30-OAc), 4.61 (1H, m, 3a-H), 5.42 (1H, d, J=4.2 Hz,6-H), 8.13 (1H, s, 3α-H), 8.42 (1H, s, 5′-H) and 10.12 (1H, s, 16-CHO).Analysis calculated for C₂₄H₃₁O₃N₃: C, 70.37; H, 7.63; N, 10.27 Found:C, 70.28; H, 7.82; N, 10.21. HRMS calcd. for C₄H₃₁O₃N₃ 409.2365, found409.2348.

Synthesis Example 3

This Example described the preparation of3β-acetoxy-17-(1H,1,2,4-triazol-1-yl)androsta-5,16-diene (Compound 5) byeither method (1) or method (2). the latter method gives a better yieldof Compound 5.

Method (1):

A mixture of the 17-triazole-16-formyl (Compound 4) (450 mg, 1.22 mmol)in dry toluene (15 ml) and Wilkinson's catalyst (1.04 g, 1.253 mmol,1.025 equiv.) was refluxed under N₂ for 2 h. The reaction mixture wasconcentrated to give a brown residue. This was treated with CH₂Cl₂(saturated with NH₃), and the resulting yellow by-product was filtered;the filtrate concentrated to give a light yellow solid (375 mg) whichwas subjected to chromatography over silica gel. Elution with CH₂Cl₂(saturated with NH₃) afforded Compound 5 (300 mg, 72%); m.p. 187-190° C.(from hexane/EtOAc). ¹H NMR (300 MHz, CDCl₃): δ 1.08 (3H, s, 18-Me),1.10 (3H, s, 19-Me), 2.04 (3H, s, 3β-OAc), 4.62 (1H, m, 3α-H), 5.42 (1H,d, J=4.5 Hz, 6-H), 5.96 (1H, s, 16-H), 7.99 (1H, s, 3′-H) and 8.26 (1H,s, 5′-H). Analysis calculated for C₂₃H₃₁O₂N₃: C, 72.40; H, 8.19; N,11.02. Found: C, 72.30; H, 8.16; N, 11.00. HRMS calcd. for C₂₃H₃₁O₂N₃381.2416, found 381.2406.

Method (2):

A mixture of bis(triphenylphosphine)rhodium(1) carbonyl chloride (338mg, 0.489 mmol) and 1, 3-bis (diphenylphosphino) propane (440 mg, 1.065mmol) in dry xylenes (40 ml) was stirred at 80° C. under N₂ for 15 minwhen a fine yellow precipitate formed.3β-Acetoxy-17-(1H-1,2,4-triazol-1-yl)-16-formylandrosta-5,16-diene(Compound 4), 2.0 g, 4.89 mmol) was added, and the mixture was refluxedunder N₂ for 15 h; then cooled, and concentrated under reduced pressure.The crude product was dissolved in EtOAc (200 ml) and filtered through a4.0 cm pad of silica gel (70-230 mesh). The silica was washed with EtOAc(2×200 ml), and the combined filtrates were evaporated to give the crudeproduct. This was purified by FCC (silica gel, pet. ether/EtOAc/Et₃N,(7.7:2:0.3)) to give Compound 5 (1.63 g, 87.6%). Spectroscopic andanalytical data were the same as given in Method 1 above.

Synthesis Example 4

This Example describes the preparation of 3β-hydroxy-17-(1H,1,2,4-triazol-1-yl)androsta-5,16-diene (Compound 6), VN/63-1.

The acetate of Compound 5 (150 mg) in dry methanol (2.0 ml) under N₂ wastreated with 10% methanolic KOH (1.0 ml). The mixture was stirred atroom temperature for 1 h, and then concentrated under reduced pressureto a volume of about 1.0 ml. It was diluted with ice-cold water (20 ml),the resulting precipitate was washed (H₂O), dried to give Compound 6(120 mg, 90%); m.p. 185-189° C. (decomp.). ¹H NMR (300 MHz, CDCl₃): δ1.07 (3H, s, 18-Me), 1.10 (3H, s, 19-Me), 3.55 (1H, m, 3a-H), 5.39 (1H,d, J=4.8 Hz, 6-H), 5.96 (1H, s, 16-H), 7.99 (1H, s, 3′-H) and 8.26 (1H,s, 51-H). Analysis calculated for C₂₁H₂₉ON₃: C, 74.29; H, 8.83; N,12.38. Found: C, 74.20; H, 8.63; N, 12.34. HRMS calcd. for C₂₁H₂₉ON₃339.2311, found 339.2297.

Synthesis Example 5

This Example describes the preparation of 17-(1H,1,2,4-triazol-1-yl)androsta-4,16-diene-3-one (Compound 7), VN/107-1.

From a mixture of3β-hydroxy-17-(1H-1,2,4-triazol-1-yl)-androsta-5,16-diene (Compound 6)(250 mg, 0.7381 mmol), 1-methyl-4-piperidone (1.18 ml) and toluene (20ml) was distilled off about 4.0 ml. Aluminum isopropoxide (253 mg 1.241mmol) was then added and the mixture was ref luxed under N₂ for 4 h.After cooling, the mixture was diluted with EtOAc (30 ml), washedsuccessively with 5.0% aq. NaHCO₃ (×3) and brine (×2), and then dried(Na₂SO₄). The solvent was evaporated and the crude product was purifiedby FCC (silica gel, CH₂Cl₂/EtOH, (30:1)) to give Compound 7 (200 mg,80.5%), mp 247-250° C. ¹H NMR (300 MHz, CDCl₃): δ 1.13 (3H, s, 18-Me),1.24 (3H, s, 19-Me), 5.76 (1H, s, 16-H), 5.95 (1H, s, 4-H), 8.00 (1H, s,3′-H), and 8.26 (1H, s, 5′-H). Analysis calculated for C₂₁H₂₇ON₃:C,74.73; H, 8.07; N, 12.46. Found: C, 74.54; H, 8.00; N, 12.50.

Synthesis Example 6

This Example describes the preparation of3β-hydroxy-17β-(1H-1,2,4-triazol-1-yl)androst-5-ene (Compound 8),VN/111-1.

A mixture of Compound 6 (200 mg, 0.590 mmol), hydrazine hydrate (0.57ml, 1.77 mmol), and acetic acid (0.35 ml) in EtOH (20 ml) was heated at80° C. while a stream of air was passed through the solution for 6 h.The reaction mixture was concentrated to about 10 ml and after cooling,it was diluted with EtOAc (30 ml) followed by washing with saturatedaqueous NaHCO₃ (10 ml×2), and brine (10 ml×2), dried (Na₂SO₄) andconcentrated to give a crude product (190 mg). This was purified by FCC(silica gel, CH₂Cl₂/EtOH, (30:1)) to give the Compound 8 (150 mg,10-74.6%), mp 246-248° C. ¹H NMR (300 MHz, CDCl₃): δ 0.56 (3H, s,18-Me), 1.01 (3H, s, 19-Me), 3.53 (1H, m, 3α-H), 4.19 (1H, t, J 9.6 Hz,17a-H), 5.37 (1H, d, J=5.2 Hz, 6-H), 7.93 (1H, s, 3′-H) and 8.10 (1H, s,5′-H). Analysis calculated for C₂₁H₃₁ON₃: C, 73.85; H, 9.16; N, 12.31.Found: C, 73.75; H, 9.40; N, 12.28.

Synthesis Example 7

This Example describes the preparation of3β-acetoxy-17-(1H-imidazol-1-yl) -16-formylandrosta-5, 16-diene(Compound 9).

A mixture of 3β-acetoxy-17-chloro-16-formylandrosta-5,16-diene (Compound3) (500 mg, 1.329 mmol), imidazole (136 mg, 2.0 mmol) and K₂CO₃ (551 mg,3.99 mmol) in dry DMF was heated at about 80° C. under N₂ for 2 h. Aftercooling to room temperature, the reaction mixture was poured ontoice-cold water (100 ml), and the resulting white precipitate wasfiltered, washed with water, and dried to give a white solid. This wastitrated with boiling mixture of hexane/EtOAc to give Compound 9 (520mg, 92%), mp 218-220° C. ¹H NMR (300 MHz, CDCl₃): δ 1.08 (6H, s, 18 and19-Me), 2.04 (3H, s, 3f-OAc), 4.61 (1H, m, 3α-H), 5.42 (1H, d, J=4.8 Hz,6-H), 7.11 (1H, s, 4′-H), 7.23 (1H, s, 5′-H), 7.63 (1H, s, 2′-H) and9.74 (1H, s, 16-CHO). Analysis calculated for C₂₅H₃₂O₃N₂: C, 73.49; H,7.90; N, 6.88; Found: C, 73.34; H, 8.05; N, 6.65. HRMS calcd. forC₂₅H₃₂O₃N₂ 408.2413, found 408.2426.

Synthesis Example 8

This Example describes the preparation of 3β-acetoxy-17-(1H-imidazol-1-yl) androsta-5,16-diene (Compound 10).

A solution of3β-acetoxy-17-(1H-imidazol-1-yl)-16-formylandrosta-5,16-diene (Compound9) (4.0 g, 9.8 mmol) in dry benzonitrile (40 ml) was refluxed in thepresence of 10% palladium on activated charcoal (2.0 g, i.e., 50% weightof the 16-formyl azole) for 3.5 h. After cooling to room temperature,the catalyst was removed by filtration through a Celite pad. Thefiltrate was evaporated and the residue was purified by FCC (silica gel,pet. ether/EtOAc/Et₃N, (6:4:0.3)) to give Compound 11 (2.72 g, 73.2%);mp 138-140° C. ¹H NMR (300 MHz, CDCl₃): δ 1.0 (3H, s, 18-Me), 1.07 (3H,s, 19-Me), 2.04 (3H, s, 3f-OAc), 4.60 (1H, m, 3α-H), 5.41 (1H, s, 6-H),5.68 (1H, s, 16-H), 7.02 (1H, s, 4′-H), 7.08 (1H, s, 5′-H), and 7.60(1H, s, 2′-H). Analysis calculated for C₂₅H₃₂O₃N₂: C, 73.49; H, 7.90; N,6.88; Found: C, 73.34; H, 8.05; N, 6.65. HRMS calcd. for C₂₅H₃₂O₃N₂408.2413, found 408.2426.

Synthesis Example 9

This Example describes the preparation of 3β-hydroxy-17-(1H-imidazol-1-yl) androsta-5,16-diene (Compound 11), VN/85-1.

The method followed that described in Synthesis Example 4, but using3,6-acetoxy-17-(1H-imidazol-1-yl) -androsta-5,16-diene (Compound 10)(2.72 g, 7.16 mmol) in methanol (30 ml), 10% methanolic KOH (17 ml), andthe mixture was stirred at room temp under N₂ for 2 h. Following theconventional workup gave rise to Compound 11 (2.34 g, 95%), mp 220-223°C. ¹H NMR (300 MHz, CDCl₃): δ 1.01 (3H, s, 18-Me), 1.06 (3H, s, 19-Me),3.53 (1H, m, 3α-H), 5.39 (1H, d, J=5 Hz, 6-H), 5.69 (1H, s, 16-H), 7.08(2H, br. s, 4′ and 5′-H), and 7.64 (1H, s, 2′-H). Analysis calculatedfor C₂₂H₃₀ON₂: C, 78.05; H, 8.94; N, 8.28; Found: C, 78.02; H, 9.00; N,8.22. HRMS calcd. for C₂₂H₃₀ON₂ 338.2358, found 338.2361.

Synthesis Example 10

This Example describes the preparation of17-(1H-imidazol-1-yl)androsta-4,16-diene-3-one (Compound 12), VN/108-1.

The method followed that described in Synthesis Example 5, but using3,-hydroxy-17-(1H-imidazol-1-yl)-androsta-5,16-diene (Compound 11) (200mg, 0.59 mmol). Purification of the crude product by FCC (silica gel,CH₂Cl₂/EtOH, (40:1)) gave Compound 12 (150 mg, 75.4%), mp 147-150° C. ¹HNMR (300 MHz, CDCl₃): δ 1.03 (3H, s, 18 -Me), 1.23 (3H, s, 19-Me), 5.69(1H, s, 6-H), 5.76 (1H, s, 16- H), 7.10 (2H, br. s, 4′ and 5′-H), and7.63 (1H, s, 2′-H). Analysis calculated for C₂₂H₂₃ON₂: C, 78.52; H,8.39; N, 8.33; Found: C, 78.30; H, 8.42; N, 8.23.

Synthesis Example 11

This Example describes the preparation of3β-Hydroxy-17β-(1H-imidazol-1-yl)androst-5-ene (Compound 13), VN/112-1.

The method followed that described for Synthesis Example 6, but using3β-hydroxy-17-(1H-imidazol-1-yl)androsta-5,16-diene (Compound 11) (170mg, 0.505 mmol) and after purification by FCC (silica gel,CH₂Cl₂/EtOAc/Et₃N, (7.7:2.0:0.3)) gave Compound 13 (110 mg, 64.3%), mp255-258° C. ¹H NMR (300 MHz, CDCl₃): δ 0.58 (3H, s, 18-Me), 1.01 (3H, s,19-Me), 3.53 (1H, m, 3α-H), 3.98 (1H, t, J=9.8 Hz, 17α-H), 5.38 (1H, d,J=5.4 Hz, 6-H), 6.96 (1H, s, 4′-H), 7.04 (1H, s, 5′-H), and 7.54 (1H, s,2′-H). Analysis calculated for C₂₂H₃₂ON₂: C, 77.59; H, 9.48; N, 8.23;Found: C, 77.55; H, 9.40; N, 8.31.

Synthesis Example 12

This Example describes the preparation of17β-(1H-imidazol-1-yl)androst-4-ene-3-one (Compound 14), VN/113-1.

The method followed that described for Synthesis Example 5, but using3β-Hydroxy-17β-(1H-imidazol-1-yl) androst-5-ene (Compound 13) (60 mg,0.1765 mmol). Purification of the crude product by FCC (silica gel,CH₂Cl₂/EtOH, (30:1)) gave Compound 12 (43 mg, 72%), mp 196-198° C. ¹HNMR (300 MHz, CDCl₃): δ 0.61 (3H, s, 18-Me), 1.19 (3H, s, 19-Me), 3.97(1H, t, J=9.6 Hz, 17α-H), 5.75 (1H, s, 6-H), 6.97 (1H, s, 4′-H)07 (2H,s, 5′-H), and 7.57 (1H, s, 2′-H). Analysis calculated for C₂₂H₃₀ON₂: C,78.05; H, 8.94; N, 8.23; Found: C, 78.10; H, 8.90; N, 8.21.

Synthesis Example 13

This Example describes the reaction of3β-acetoxy-17-chloro-16-formylandrosta-5, 16-diene (Compound 3) with1H-1,2,3-Triazole and K₂CO₃ to give3β-acetoxy-17-(2H-1,2,3-triazol-2-yl)-16-formylandrosta-5,16-diene(Compound 15) and 3β-acetoxy-17- (1-H-1, 2, 3-triazol-1-yl)-16-formylandrosta-5, 16-diene (Compound 16), respectively.

A mixture of 3β-acetoxy-17-chloro-16-formylandrosta-5,16-diene (Compound3) (2.0 g, 5.23 mmol), 1H-1,2,3-triazole (552 mg, 7.98 mmol) and K₂CO₃(2.20 g, 15.95 mmol) in dry DMF (40 ml) was heated at 80° C. under N₂atmosphere for 2 h. After cooling to room temperature, the reactionmixture was poured onto ice-water (400 ml), and the resultingprecipitate was filtered, washed with water, and dried to give a dirtywhite solid. This was subjected to flash chromatography, and on elutionwith pet. ether/EtOAc/Et₃N, (6.7:3:0.3), gave firstly3β-acetoxy-17-(2H-1,2,3-triazol-2-yl)-16-formylandrosta-5,16-diene(Compound 15) (684 mg, 28%), mp 145-148° C. ¹H NMR (300 MHz, CDCl₃): δ1.09 (3H, s, 18-Me), 1.26 (3H, s, 19-Me), 2.04 (3H, s, 3β,-OAc), 4.61(1H, m, 3α-H), 5.42 (1H, d, J=4.2 Hz, 6-H), 7.85 (2H, s, 4′ and 5′-H)and 10.55 (1H, s, 16-CHO). Analysis calculated for C₂₄H₃₁O₃N₃: C, 70.37;H, 7.63; N, 10.27. Found: C, 70.30; H, 7.95; N, 9.87. Further elutionwith pet. ether/EtOAc/Et₃N, (6:4:0.3) gave3β-acetoxy-17-(1H-1,2,3-triazol-1-yl) -16-formylandrosta-5,16-diene(Compound 16) (1.48 g, 62%), mp 215-217° C. ¹H NMR (300 MHz, CDCl₃): δ1.08 (3H, s, 18-Me), 1.18 (3H, s, 19-Me), 2.04 (3H, s, 3β-OAc), 4.63(1H, m, 3α-H), 5.43 (1H, d, J=4.2 Hz, 6-H), 7.85 (2H, s, 4′ and 5′-H)and 9.94 (1H, s, 16-CHO). Analysis calculated for C₂₄H₃₁O₃N₃: C, 70.37;H, 7.63; N, 10.27. Found: C, 70.37; H, 7.86; N, 10.10.

Synthesis Example 14

This Example describes the preparation of3β-acetoxy-17-(2H-1,2,3-triazol-2-yl) androsta-5, 16-diene (Compound17).

A mixture of3β-acetoxy-17-(2H-1,2,3-triazol-2-yl)-16-formylandrosta-5,16-diene(Compound 16) (140 mg, 0.342 mmol) in dry toluene (6.0 ml) and tris(triphenylphoshpine) rhodium (1) chloride (Wilkinson's catalyst,; 332 mg0.351 mmol) was ref luxed under N₂ for 5 h. After cooling to roomtemperature, EtOH (12 ml) was added and on further cooling at approx. 0°C., the yellow precipitate of bis (triphenylphosphine)carbonylchlororhodium(1) formed.

Following filtration, the filtrate was concentrated to give the crudeproduct. This was purified by FCC (silica gel, pet. ether/EtOAc, (15:1))to give Compound 17, a white solid (120 mg, 92%), mp 154-155° C. ¹H NMR(300 MHz, CDCl₃): δ 1.09 (3H, s, 18-Me), 1.14 (3H, s, 19-Me), 2.04 (3H,s, 3,B-OAc), 4.60 (1H, m, 3α-H), 5.42 (1H, d, J=4.2 Hz, 6-H), 6.17 (1H,br. s, 16-H) and 7.68 (21, s, 4′ and 5′-H). Analysis calculated forC₂₃H₃₁O₂N₃: C, 72.40; H, 8.19; N, 11.02. Found: C, 72.16; H, 8.32; N,10.90.

Synthesis Example 15

This Example describes the preparation of3β-hydroxy-17-(2H-1,2,3-triazol-2-yl)androsta-5,16-diene (Compound 18),VN/90-1.

The method followed that described in Synthesis Example 4, but using3β-acetoxy-17-(2H-1,2,3-triazol-2-yl)androsta-5,16-diene (Compound 17)(110 mg, 0.289 mmol). Purification of the crude product by FCC (silicagel, pet. Ether/EtoAc, (3:1)) gave Compound 18 (95 mg, 97.1%) which wascrystallized from hexane/EtOAc, mp 176-177° C. ¹H NMR (300 MHz, CDCl₃):δ 1.09 (3H, s, 18-Me), 1.15 (3H, s, 19-Me), 3.54 (1H, m, 3α-H), 5.39(1H, d, J=5.1 Hz, 6-H), 6.17 (1H, s, 16-H) and 7.68 (2H, s, 4′ and5′-H). Analysis calculated for C₂₁H₂₉ON₃: C, 74.29; H, 8.83; N, 12.38.Found: C, 72.16; H, 8.32; N, 10.90.

Synthesis Example 16

This Example describes the preparation of3β-acetoxy-17-(1H-1,2,3-triazol-1-yl)androsta-5,16-diene (Compound 19).

The method followed that described for Synthesis Example 3, Method 2,but using303-acetoxy-17-(1H-1,2,3-triazol-1-yl)-16-formylandrosta-5,16-diene(Compound 16) (2.0 g, 4.89 mmol). Purification of the crude product byFCC (silica gel, pet. ether/EtOAc/Et₃N, (7.7:2:0.3)) gave Compound 19(1.67 g, 89.9%), mp 158-160° C. ¹H NMR (300 MHz, CDCl₃): δ 1.09 (3H, s,18-Me), 1.14 (3H, s, 19-Me), 2.04 (3H, s, 3,8-OAc), 4.60 (1H, m, 3a-H),5.40 (1H, d, J=4.2 Hz, 6-H), 5.98 (1H, br. s, 16-H) and 7.73 (2H, s, 4′and 5′-H). Analysis calculated for C₂₃H₃₁O₂N₃: C, 72.40; H, 8.19; N,11.02. Found: C, 72.20; H, 8.21; N, 11.00.

Synthesis Example 17

This Example describes the preparation of3β-hydroxy-17-(1H-1,2,3-triazol-1-yl)androsta-5,16-diene (Compound 20),VN/87-1.

The method followed that described in Synthesis Example 4, but using3β-acetoxy-17-(1H-1,2,3-triazol-1-yl)androsta-5,16-diene (Compound 19)(1.5 g, 3.94 mmol). The product was recrystallized from EtOAc/MeOH togive Compound 19 (1.20 g, 90%), mp 220-224° C. ¹H NMR (300 MHz, CDCl₃):δ 1.08 (3H, s, 18-Me), 1.14 (3H, s, 19-Me), 3.54 (1H, m, 3α-H), 5.39(1H, d, J=4.8 Hz, 6-H), 5.97 (1H, s, 16-H) and 7.72 (2H, s, 4′ and5′-H). Analysis calculated for C₂₁H₂₉ON₃: C, 74.29; H, 8.83; N, 12.38.Found: C, 74.10; H, 8.70; N, 12.15.

Synthesis Example 18

This Example describes the preparation of17-(1H-1,2,3-triazol-1-yl)androsta-4,16-diene-3-one (Compound 21),VN/109-1.

The method followed that described in Synthesis Example 5, but using3β-hydroxy-17-(1H-1,2,3-triazol-1-yl)-androsta-5,16-diene (Compound 20)(400 mg, 1.18 mmol). Purification of the crude product by FCC (silicagel, CH₂Cl₂/EtOH, (30:1)) gave Compound 21 (358 mg, 90%), mp 118-120° C.¹H NMR (300 MHz, CDCl₃): δ 1.17 (3H, s, 18-Me), 1.25 (3H, s, 19-Me),5.76 (1H, s, 16-H), 5.95 (1H, s, 4-H), 7.73 (1H, s, 5′-H), and 7.74 (1H,s, 4′-H). Analysis calculated for C₂₁H₂₇ON₃:C, 74.73; H, 8.07; N, 12.46.Found: C, 74.65; H, 8.11; N, 12.34.

Synthesis Example 19

This Example describes the preparation of 3β-acetoxy-17-(2H-tetrazol-2-yl) -16-formylandrosta-5, 16-diene (Compound 22) and3,-Acetoxy-17-(1H-tetrazol-1-yl)-16-formylandrosta-5,16-diene (Compound23).

The method followed that described for Synthesis Example 7, but using3β-acetoxy-17-chloro-16-formylandrosta-5,16-diene (Compound 3) (0.5 g,1.329 mmol), 1H-tetrazole (187 mg, 1.59 mmol) and Li₂CO₃ (287 mg, 3.94mmol) gave a crude product (520 mg). Flash column chromatography onelution with pet. ether/EtOAc, (5:1), gave firstly3β-acetoxy-17-(2H-tetrazol-2-yl)-16-formylandrosta-5,16-diene (Compound22) (92 mg, 28.2%), mp 170-172° C. (decomp.). ¹H NMR (300 MHz, CDCl₃): δ1.1 (3H, s, 18-Me), 1.29 (3H, s, 19-Me), 2.04 (3H, s, 36-OAc), 4.62 (1H,m, 3α-H), 5.42 (1H, d, J=4.2 Hz, 6-H), 8.68 (1H, s, 5′-H) and 10.46 (1H,s, 16-CHO). Analysis calculated for C₃H₃%N4: C, 67.28; H, 7.37; N,13.65. Found: C, 67.15; H, 7.61; N, 13.45. Further elution with p.ether/EtOAc/Et₃N (7:3:0.3) gave 3β-acetoxy-17-(1H-tetrazol-1-yl)-16-formylandrosta-5, 16-diene (Compound 23) (146 mg, 44.6%), mp196-198° C. (decomp.). ¹H NMR (300 MHz, CDCl₃): δ 1.09 (3H, s, 18-Me),1.20 (3H, s, 19-Me), 2.04 (3H, s, 3f-OAc), 4.62 (1H, m, 1S 3α-H), 5.43(1H, d, J=4.8 Hz, 6-H), 8.93 (1H, s, 5′-H) and 9.92 (1H, s, 16-CHO).Analysis calculated for C₂₃H₃₀O₃N₄: C, 67.28; H, 7.37; N, 13.65. Found:C, 67.05; H, 7.65; N, 13.45.

Synthesis Example 20

This Example describes the preparation of3β-acetoxy-17-(2H-tetrazol-2-yl)androsta-5,16-diene (Compound 24).

The method followed that described for Synthesis Example 14, but using3β-acetoxy-17-(2H-tetrazol-2-yl)-16-formylandrosta-5,16-diene (Compound22) (124 mg, 0.302 mmol). Purification of the crude product by FCC(silica gel, pet. ether/EtOAc (10:1)) gave Compound 24 (61 mg, 52.8%),mp 155-156° C. ¹H NMR (300 MHz, CDCl₃): δ 1.10 (3H, s, 18-Me), 1.17 (3H,s, 19-Me), 2.04 (3H, s, 3β-OAc), 4.62 (1H, m, 3a-H), 5.42 (1H, d,J=4.85Hz, 6-H), 6.46 (1H, s, 16-H) and 8.52 (1H, s, 5′-H). Analysiscalculated for C₂₂H₃₀O₂N₄: C, 69.07; H, 7.91; N, 14.65. Found: C, 69.01;H, 8.00; N, 14.45.

Synthesis Example 21

This Example describes the preparation of3β-hydroxy-17-(2H-tetrazol-2-yl)androsta-5, 16-diene (Compound 25),VN/96-1.

The method followed that described for Synthesis Example 4, but using3β-acetoxy-17-(2H-tetrazol-2-yl)androsta-5,16-diene (Compound 24) (51mg, 0.134 mmol). Recrystallization of the product from hexane/EtOAc gaveCompound 25 (42 mg, 88%), mp 195-198° C. NMR (300 MHz, CDCl₃): δ 1.09(3H, s, 18-Me), 1.17 (3H, s, 19-Me), 3.55 (1H, m, 3α-H), 5.43 (1H, d,J=5.2Hz, 6-H), 6.46 (1H, s, 16-H) and 8.53 (1H, s, 5′-H). Analysiscalculated for C₂₀H₂₈ON₄: C, 70.54; H, 8.29; N, 16.46. Found: C, 70.51;H, 8.25; N, 16.50.

Synthesis Example 22

This Example describes the preparation of3β-acetoxy-17-(1H-tetrazol-1-yl)androsta-5,16-diene (Compound 26).

The method followed that described for Synthesis Example 14, but using3β-acetoxy-17-(1H-tetrazol-1-yl)-16-formylandrosta-5,16-diene (Compound23) (140 mg, 0.3415 mmol). Purification of the crude product by FCC(silica gel, pet. ether/EtOAc (3:1)) gave Compound 26 (45 mg, 34.5%); ¹HNMR (300 MHz, CDCl₃): δ 1.08 (3H, s, 18-Me), 1.20 (3H, s, 19-Me), 2.04(3H, s, 3,-OAc), 4.62 (1H, m, 3α-H), 5.42 (1H, d, J=4.6Hz, 6-H), 6.00(1H, s, 16-H) and 8.93 (1H, s, 5′-H). This compound was not particularlystable at room temp. (TLC evidence) and was used for the subsequentreaction without further characterization.

Synthesis Example 23

This Example describes the preparation of 3β-hydroxy-17-(1H-tetrazol-1-yl) androsta-5,16-diene (Compound 27), VN/95-1.

The method followed that described for Synthesis Example 4, but using3β-acetoxy-17-(1H-tetrazol-1-yl)androsta-5,16-diene (Compound 26) (36mg, 0.094 mmol). Recrystallization of the product from hexane/EtOAc gaveCompound 27 (28 mg, 87.4%), mp 200-204° C. (decomp.). NMR (300 MHz,CDCl₃): δ 1.06 (3H, s, 18-Me), 1.11 (3H, s, 19-Me), 3.54 (1H, m, 3α-H),5.38 (1H, br. s, 6-H), 6.13 (1H, s, 16-H) and 8.73 (1H, s, 5′-H).Analysis calculated for C₂₀H₂₈ON₄: C, 70.54; H, 8.29; N, 16.46. Found:C, 70.55; H, 8.20; N, 16.42.

Synthesis Example 24

This Example describes the preparation of3β-acetoxy-17-(1H-pyrazol-1-yl) androsta-5,16-diene (Compound 28) and3,-acetoxy-17α-(1H-pyrazol-1-yl)androsta-5-ene (Compound 29).

(a) Reaction of 3β-acetoxy-17-chloro-16-formylandrosta-5,16-diene(Compound 3) (0.5 g, 1.329 mmol), pyrazole (136 mg, 1.994 mmol) andK₂CO₃ (551 mg, 3.99 mmol) as described for Synthesis Example 7 after FCC(silica gel, pet. ether/EtOAc, (4:1)) gave a mixture (341 mg, approx.3:1) of 3β-acetoxy-17-(1H-pyrazol-1-yl) -16-formylandrosta-5,16-dieneand 3β-acetoxy-17- (1H-pyrazol-1-yl) androsta-5-ene. This mixtureresisted separation by chromatography.

(b) The above mixture (330 mg) was subjected to the decarbonylationreaction as described for Synthesis Example 14 to give a crude product(350 mg). Flash column chromatography on elution with pet. ether/EtOAc,(15:1), gave firstly 3β-acetoxy-17- (1H-pyrazol-1-yl)androsta-5,16-diene(Compound 28) (123 mg, 35%), mp 159-161° C. ¹H NMR (300 MHz, CDCl₃): δ1.08 (3H, s, 18-Me), 1.11 (3H, s, 19- Me), 2.04 (3H, s, 38-OAc), 4.62(1H, m, 3α-H), 5.42 (1H, d, J=5.1 Hz, 6-H), 5.77 (1H, s, 16-H), 6.32(1H, s, 4′-H), 7.60 (1H, s, 3′-H) and 7.63 (1H, d, J=2.4 Hz, 5′-H).Analysis calculated for C₂₄H₃₂O₂N₂: C, 75.74; H, 8.48; N, 7.37. Found:C, 75.94; H, 8.51; N, 7.33. Further elution with pet. ether/EtOAc, (5:1)gave 3β-acetoxy-17α-(1H-pyrazol-1-yl)androst-5-ene (Compound 29) (93 mg,30%), mp 238-240° C. ¹H NMR (300 MHz, CDCl₃): δ 0.97 (3H, s, 18-Me),1.08 (3H, s, 19-Me), 2.04 (3H, s, 3β-OAc), 3.24 (1H, dd, J=4.8 Hz,J₂=15.6 Hz, 17β-H), 4.62 (11H, m, 3α-H), 5.43 (1H, d, J=5.1 Hz, 6-H),6.45 (1H, s, 4′-H), 7.70 (1H, d, J=2.4 Hz, 3′-H) and 7.77 (1H, d, J=4.8Hz, 5′-H). Analysis calculated for C₂₄H₃₄O₂N₂: C, 75.34; H, 8.96; N,7.33. Found: C, 75.24; H, 8.90; N, 7.30.

Synthesis Example 25

This Example describes the preparation of3β-hydroxy-17-(1H-pyrazol-1-yl)androsta-5,16-diene (Compound 30),VN/97-1.

This method followed that described for Synthesis Example 4, but using3β-acetoxy-17-(1H-pyrazol-1-yl)androsta-5,16-diene (Compound 25) (100mg, 0.236 mmol). Recrystallization of the product from hexane/EtOAc gaveCompound 30 (85.8 mg, 95.6%), mp 197-199° C.; ¹H NMR (300 MHz, CDCl₃): δ1.06 (3H, s, 18-Me), 1.09 (3H, s, 19-Me), 3.56 (1H, m, 3α-H), 5.39 (1H,s, 6-H), 5.78 (1H, s, 16-H), 6.31 (1H, s, 4′-H), 7.59 (1H, s, 3′-H) and7.62 (1H, s, 5′-H). Analysis calculated for C₂₂H₃₂ON₂: C, 77.59; H,9.48; N, 8.32. Found: C, 77.66; H, 9.59; N, 8.21.

Example 1

Evaluation of 17-Azolyl Steroids as Inhibitors of Testicular Human andRat 17α-hydroxylase/C_(17,20)-lyase (17α-lyase) in vitro

The potency as inhibitors of P450_(17α) of the 17-azolyl steroidsobtained in the above Synthesis Examples was evaluated in human and rattesticular microsomes.

Human testicular microsomes were prepared from human testes (obtainedfrom untreated prostatic cancer patients undergoing orchidectomy in theUniversity of Maryland Hospital and Veterans Hospital), as described inLi et al, The Prostate, 26:140-150 (1995).

Rat testicular microsomes were prepared from the testes of adultSprague-Dawley rats (Charles River Laboratories, weight 200-250 g), asdescribed by Li et al, J. Med. Chem., 39:4335-4339 (1996).

The microsomes were stored at −70° C. until assayed. Just before use,the thawed microsomes were diluted with 0.1 M phosphate buffer (pH 7.4)to appropriate concentrations.

The protein concentration of the microsomes used in the assay wasdetermined by the method of Lowry et al, J. Biol. Chem., 193:265-275(1951).

The enzyme reaction (activity) was monitored by determination of therelease of C³H₃COOH from [21-³H₃]-17α-hydroxypregnenolone duringcleavage of the 30 C-21 side-chain in the conversion todehydroepiandrosterone (DHEA) as described by Njar et al, Steroids,62:468-473 (1997). This assay measures only the lyase activity of theP450_(17α) enzyme. This assay is comparable to the HPLC assay procedure(which utilizes [7-³H]-pregnenolone as substrate), and measures both thehydroxylase and lyase activities of the enzyme.

The results are presented in Tables 1 and 2 below:

TABLE 1 The Inhibition of Human P450_(17α) by Δ¹⁶-17-Azolyl SteroidsCompound^(a) % Inhibition^(b)  6 (VN/63-1) 60 17 (VN/85-1) 97 18(VN/90-1) N.I.^(c) 20 (VN/87-1) 94 25 (VN/96-1) N.I.^(c) 27 (VN/95-1)N.I.^(c) 30 (VN/97-1) 40 for comparison Ketoconazole 67 ^(a)Eachinhibitor concentration was 150 nM. ^(b)Concentration of substrate,17α-hydroxypregnenolone = 10 μM. ^(c)N.I. = No inhibition atconcentration of 150 nM. All values are the mean of two determinations.

TABLE 2 Inhibitory Potency of Δ¹⁶-17-Azolyl Steroids Towards Human andRat P450_(17α) and Human Steroid 5α-Reductase Human P450_(17α) RatP450_(17α) 5α-Reductase Compound IC₅₀ (nM)^(a) K_(i) (nM)^(b,c) IC₅₀(nM)^(a) IC₅₀ (nM)^(a)  6 (VN/63-1) 90 ± 14 23 26 ± 13 ˜160,000  7(VN/107-1) 55 ± 11 41 11 ± 3  152 ± 10   8 (VN/111-1) 219 ± 21  — — — 11(VN/85-1) 8 ± 1 1.2 9 ± 2 ˜400,000 12 (VN/108-1) 7 ± 1 1.9   8 ± 0.7 142± 5  13 (VN/112-1) 62 ± 2  — — — 14 (VN/113-1) 36 ± 9  — — 765 ± 100 20(VN/87-1) 13 ± 1  1.4  10 ± 0.4  ˜10,000 21 (VN/109-1) 19 ± 1  8 9 ± 2198 ± 33  for comparison Ketoconazole 78 ± 3  38 209 ± 17  — Finasteride— — — 33 ± 2  ^(a)Mean ± SDM of at least two experiments. ^(b)K_(i)values were determined as described herein. ^(c)K_(m) for substrate,17α-hydroxypregnenolone = 560 nM.

In all experiments, the blank activity ranged from 1-5% of the controlactivity. IC₅₀ values for inhibitors were calculated from the linearregression line in the plot of logit of lyase activity versus log ofinhibitor concentration. K_(i) values were also determined from assaysas described by Njar et al (1997), supra. Each inhibitor was examined atthree concentrations. Data from the various assays were used to obtainLineweaver-Burk plots and from replots of slopes versus inhibitorconcentration (FIG. 2B), K_(i) values were obtained and the K_(m) for17α-hydroxypregnenolone (substrate) was also determined (Table 2).

In order to estimate the inhibitor potency of the compounds of thepresent invention, the tritiated substrate, a NADPH generating systemand microsomes were incubated at 34° C. in O₂ in the presence or absenceof the inhibitor. The reaction was usually monitored for 60 min duringwhich time the formation of [³H]-acetic acid, and thus DHEA was linear.The percentage inhibition data for the initial target compounds of thisstudy are presented in Table 1 and highlights that 2H-1,2,3-triazole(Compound 18, VN/90-1) and the two tetrazole regioisomers (Compound 25,VN/96-1; and Compound 27, VN/95-1) were non-inhibitory, while the1H-pyrazole (Compound 30, VN/97-1) was a moderate inhibitor. By contrastthe 1H-1,2,4-triazole (Compound 6, VN/63-1), 1H-imidazole (Compound 11,VN/85-1) and 1H-1,2,3-triazole (Compound 20, VN/87-1) were potentinhibitors of the enzyme. Ketoconazole also showed strong inhibition.Given that these Δ¹⁶-17-azole compounds of Table 1 are structurallysimilar, (i.e., they all possess the Δ⁵-3β-ol functionality) thestriking difference in the inhibitory properties observed may be due tothe differences in their basicities, a property imposed by the inherentdifferent electronic character of each of the azole heterocycles. Inaddition, the presence of a nitrogen atom at either the 3′ or 4′position seems important for potent inhibition of the enzyme.

Following the initial screening assays, Compounds 6, 11 and 20 togetherwith their corresponding Δ⁴-3-one counterparts, Compounds 7 (VN/107-1),12 (VN/108-1) and 21 (VN/109-1), respectively, were evaluated further todetermine firstly, their IC₅₀ values and then their apparent K_(i)values (from Lineweaver-Burk plots, e.g., FIG. 2A). These values arepresented in Table 2. All six 17-azoles are excellent noncompetitiveinhibitors of P450_(17α) as shown in the example in FIG. 2A. The natureof inhibition kinetics exhibited by these compounds was that in whichthe V_(max) was decreased, but the apparent K_(m) was unchanged; i.e.,the intercept on the horizontal axis is the same in the absence orpresence of inhibitor. This is one of two characteristics of anoncompetitive inhibitor, and indicates destruction of the catalyticactivity of the enzyme. The other is when binding of the inhibitor and(variable) substrate are not mutually exclusive.

There was no marked difference between the inhibitory potencies of theΔ⁵-3β-ol azoles (Compounds 6, 11 and 20) with those of the correspondingΔ⁴-3-ones (Compounds 7, 12 and 21).

Three of the compounds, i.e., Compounds 7, 12 and 20 with K_(i) valuesof 1.2, 1.8 and 1.4 nM, respectively, (K_(m) of the substrate,17α-hydroxypregnenolone was 530 nM), were the most potent inhibitors,and they are indeed the most potent inhibitors of human testicularmicrosomal P450_(17α) described to date. These compounds were 20-32times more potent as P450_(17α) inhibitors when compared in the sameassay with ketoconazole (K_(i)32 38 nM). Some Δ¹⁶-17-(3-pyridyl)compounds were recently classified as the most potent inhibitors of thisenzyme (Potter et al, supra). However, three of their most potentinhibitors were 9-12 times more potent as P450_(17α) (lyase activity)inhibitors when compared in the same assay with ketoconazole (Potter etal, supra). The requirement of 16,17-double bond was also observed withthese P450_(17α) inhibitors: 17β-(1H-1,2,4-triazolyl)- and17β-(1H-imidazolyl)- compounds, Compounds 8 (VN/111-1) and 13 (VN/112-1)each exhibited diminished potency compared to the corresponding parentΔ¹⁶ compounds, Compounds 6 and 11, respectively, (Compound 6→Compound 8,IC₅₀ 90→219 nM, and Compound 11→Compound 13, IC₅₀ 8→62 nM). A similarobservation has been previously reported (Potter et al, supra; Burkhartet al, Bioorg. Med. Chem., 4:1411-1420 (1996); and Ling et al, supra)for a number of Δ¹⁶-17-heteroaryl P450_(17α) inhibitors. Conversion ofCompound 13 to the Δ⁴-3-one compound, Compound 14 resulted in a modestincrease in inhibitory activity (62→36 nM).

When the lyase reaction was monitored in the presence of variousconcentrations of the imidazole, Compound 11, a family of non-linearprogress curves were obtained in which the extent of inhibitionincreased with time (FIG. 3). This suggest that Compound 11 may be aslow-binding inhibitor (Morrison, et al, Adv. Enzymol. Relat. Areas Mol.Biol., 61:201-301 (1988)). Although the other potent inhibitors were notexamined in this assay, it is likely that they may also behave in asimilar fashion. Compound 11 appears to be the first example of aslow-binding inhibitor of cytochrome P450_(17α).

To investigate the mechanism of P450_(17α) inhibition further, theproperties (chemical nature) of the complexes formed between the1H-1,2,4-triazole, Compound 6 and imidazole, Compound 11 and a modifiedform of human P450_(17α) (Imai et al, J. Biol. Chem., 268:19681-19689(1993) were next studied using UV-VIS difference spectroscopy asdescribed by Jefcoat, Methods Enzymol., 52:258-279 (1978). Each of thesecompounds induced a Type II difference spectrum (FIG. 4), indicatingcoordination of a steroidal nitrogen (probably N-4 of the triazole ringor N-3 of the imidazole ring) to the here iron of the cytochrome P450enzyme, with formation of low spin iron. The peak positions of the Soretmaxima for the complexes with triazole, Compound 6 (422 nm) andimidazole, Compound 11 (426 nm) are in agreement with available data forthe binding of nitrogen ligands to cytochrome P450 systems; resulting incomplexes with Soret maximum at 421-430 nm (Dawson et al, J. Biol.Chem., 257:3606-3617 (1982)).

The inhibitory potency of (20R)- and (20S)-aziridinyl steroids haverecently been reported, and this stems in part from the additionalstabilization due to coordination of the heteroatom of their aziridinylring to the heme of rat P450_(17α) (Njar et al, Bioorg. Med. Chem.,4:1447-1453 (1996)). The spectroscopic data described above suggest thatthis may also be the case for the Δ¹⁶-17-azole steroids of the presentinvention. The ability of the steroidal azole nitrogen atom tocoordinate with the heme of P450_(17α) indicates that C-17 and C-20 (thesites of enzymatic hydroxylations) can be positioned in close proximityto the heme center when these substrate-like inhibitors are bound to theenzyme. Although it is not certain that these compounds bind in exactlythe same manner as the natural substrates, their high binding affinitiesmake a significantly different mode of binding unlikely. It should benoted that although two groups (Potter et al, supra; and Burkhart et al,supra) have recently reported on 17-heteroaryl steroidal inhibitors ofP450_(17α), and believe that the inhibitory property of their compoundsare due (in part) to coordination of a heteroaryl atom to the heme-ironof the enzyme complex, they are yet to provide evidence for thisphenomenon.

Before evaluating these potent inhibitors in vivo in rodent models aspotential therapeutic agents for the treatment of prostate cancer, thepotency of these inhibitors was also accessed towards the rat testicularmicrosomal P470_(17α). A comparison was made between the inhibitoryactivity, expressed as IC₅₀ values, displayed by the Δ⁵-3β-ols,Compounds 6, 11 and 20; the Δ⁴-3-one compounds, Compounds 7, 12 and 21,and ketoconazole towards P470_(17α) located in human and rat testicularmicrosomes. The results are presented in Table 2 and show that whereasthe potencies of Compounds 6, 7 and 21 each increased towards the ratenzyme by 3.5-, 5- and 2-folds, respectively, the potencies of Compounds11, 12 and 20 were unchanged, while that for ketoconazole decreased byabout 3-fold. The most potent inhibitors, Compounds 11, 12 and 20 appearto be the first examples of inhibitors that are equipotent towards thehuman as well as the rat P450_(17α) enzymes. This finding indicates thatresults from pre-clinical in vivo studies with rats are likely toreflect the clinical situation.

Example 2

Evaluation of 17-Azolyl Steroids as Inhibitors of 5α-Reductase

The effects of the compounds of the present invention and finasteride (apotent inhibitor of this enzyme) on human prostate 5α-reductase activitywas evaluated essentially as described by Li et al 1996, supra; and Kluset al, Cancer Res., 56:4956-4964 (1996).

More specifically, ethanolic solutions of [7-³H]-testosterone (600,000dpm), cold testosterone (4.8 ng), indicated inhibitors (0-200 nM) andpropylene glycol (10 μl) were added to sample tubes in duplicate. Theethanol was evaporated to dryness under a gentle stream of air. Thesamples were reconstituted in 400 μl of 0.1 M phosphate buffer (pH 7.4)containing 78 μM DTT and the NADPH generating system comprising 6.5 mMNADP; 71 mm glucose-6-phosphate, and 2.5 IU glucose-6-phosphatedehydrogenase in 100 μl of phosphate buffer, was added to each tube. Thetubes were preincubated at 37° C. for 15 min. The enzymatic reactionswere initiated by addition of human BPH microsomes (about 180 μg ofmicrosomal protein in 500 μl of phosphate buffer) in a total volume of1.0 ml, and the incubations were performed for 10 min under oxygen in ashaking water bath at 37° C. The incubations were terminated by placingthe sample tubes on ice and the addition of ether. Also, [¹⁴C]-DHT (3000dpm) and cold DHT (50 μg) were added to each tube as an internalstandard and visualization marker, respectively. The steroids wereextracted with ether and separated by TLC (chloroform:ether, 80:20) andvisualized by exposure to iodine vapor. The extracts were analyzed for³H and ¹⁴C using a liquid scintillation counter. The percentageconversion of [7-³H]-testosterone to [³H]-dihydrotestosterone wascalculated and used to determine 5α-reductase activity. IC₅₀ values weredetermined from plots of 5α-reductase activity against four differentconcentrations of the inhibitor.

The results are presented in Table 2 and highlights that the Δ⁵-3β-olcompounds (Compounds 6, 11 and 20) were poor inhibitors of the enzyme.By contrast, the corresponding Δ⁴-3-one compounds (Compounds 7, 12 and21) were potent inhibitors, being only about 4-6 times less potent thanfinasteride, a potent 5α-reductase inhibitor currently used in thetreatment of benign prostatic hyperplasia.

Example 3

The Effect of Inhibitors on Androgen-dependent Growth of Human ProstateCancer (LNCaP) Cells in vitro

The abilities of Compounds 11 and 20 to inhibit the androgen-stimulatedgrowth of LNCaP human prostatic cancer cell were examined. As previouslyreported by Klus et al, supra, 0.1 nM testosterone increased the growthof these LNCaP cells 6-fold compared to vehicle-treated cells, and 30 μMDHT stimulated proliferation 5-fold compared to control. The imidazole,Compound 11 was more effective than Compound 20 in inhibiting thetestosterone-stimulated growth of LNCaP cells, with 100% inhibitionoccurring at 1.0 and 2.5 μM, respectively. Both compounds also inhibitedDHT-induced cell growth with Compound 11 again being more effective(100% inhibition at 2.5 and 5.0 μM, respectively). Since neithercompound inhibited 5α-reductase nor was toxic to the cells in theconcentration range 0.5-5.0 μM, these results indicate that theirgrowth-inhibiting properties are due to possible anti-androgeniceffects.

In summary, the present invention describes a method for theintroduction of a variety of azolyl groups at the 17-carbon of a Δ¹⁶steroid. This enabled the synthesis of several Δ¹⁶-17-azolyl steroids ofwhich Compounds 6, 7, 11, 12, 20, and 21 proved to be powerfulinhibitors of both human and rat testicular P450_(17α). In addition, itis shown that a nitrogen of Compound 6 and 11, each coordinates to theenzyme's heme-iron. Kinetic studies allowed for classification of thesecompounds as noncompetitive inhibitors of the enzyme. Unlike mostpreviously described P450_(17α) inhibitors which show normal competitiveor noncompetitive reversible kinetics, the most potent inhibitor,Compound 11 shows an apparent slow binding behavior. Compounds 12, 20and 21 are also potent inhibitors of 5α-reductase, while Compounds 11and 20 appear to possess strong antiandrogenic effects. These dualbiological properties of some of these compounds increase their utilityin the treatment of prostate cancer.

While the invention has been described in detail, and with reference tospecific embodiments thereof, it will be apparent to one of ordinaryskill in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof.

What is claimed:
 1. A compound of general Formula (I):

wherein X represents the residue of the A, B and C rings of a6-azasteroid consisting of a 4-en-3-one or 5-en-3β-ol system; Azrepresents an azole ring attached to C-17 of the steroid via a heteronitrogen atom; and R₁ and R₂ represent a hydrogen atom or togetherrepresent a double bond.
 2. The compound of claim 1, where Az isselected from the group consisting of


3. The compound of claim 1, wherein said steroid further comprises abasic structure selected from the group consisting of Δ⁴-3-one, A⁵-3β-oland Δ¹⁴-3-one.
 4. A pharmaceutical composition comprising of at leastone compound according to claim 1; and a pharmaceutically acceptablecarrier or diluent.
 5. A method for reducing plasma levels oftestosterone and/or dihydrotestosterone (DHT) in a subject in need ofsuch treatment comprising administering to said subject at least onecompound according to claim 1 in an amount sufficient to reduce plasmalevels of testosterone and/or DTH.
 6. A method for treating benignprostatic hyperblasia in a subject in need of such treatment comprisingadministering to said subject at least one compound according to claim 1in an amount sufficient to reduce the size of the prostate.
 7. A methodfor treating prostate cancer in a subject in need of such treatmentcomprising administering to said subject at least one compound accordingto claim 1 in an amount sufficient to reduce the size of the prostatetumors.